U.S. patent application number 12/397870 was filed with the patent office on 2009-10-08 for methods and devices for bubble mitigation.
This patent application is currently assigned to Helicos BioSciences Corporation. Invention is credited to John Kepler, Alexander Watson.
Application Number | 20090249949 12/397870 |
Document ID | / |
Family ID | 41132065 |
Filed Date | 2009-10-08 |
United States Patent
Application |
20090249949 |
Kind Code |
A1 |
Kepler; John ; et
al. |
October 8, 2009 |
METHODS AND DEVICES FOR BUBBLE MITIGATION
Abstract
The invention relates to methods, systems and devices for
mitigation of bubbles in a micro-fluidic environment. For example,
the invention relates to methods, systems and devices for
mitigation of bubbles from reagents, solvents, formulations and for
improving chemical reactions in micro-fluidic systems, such as for
fluorescence detection and polynucleotide sequencing.
Inventors: |
Kepler; John; (Lexington,
MA) ; Watson; Alexander; (Gloucester, MA) |
Correspondence
Address: |
COOLEY GODWARD KRONISH LLP;ATTN: Patent Group
Suite 1100, 777 - 6th Street, NW
WASHINGTON
DC
20001
US
|
Assignee: |
Helicos BioSciences
Corporation
Cambridge
MA
|
Family ID: |
41132065 |
Appl. No.: |
12/397870 |
Filed: |
March 4, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61034140 |
Mar 5, 2008 |
|
|
|
Current U.S.
Class: |
95/44 ; 95/155;
96/6 |
Current CPC
Class: |
B01L 3/561 20130101;
B01L 3/502723 20130101; B01D 19/0042 20130101; B01D 19/0036
20130101; B01D 19/0031 20130101 |
Class at
Publication: |
95/44 ; 95/155;
96/6 |
International
Class: |
B01D 19/02 20060101
B01D019/02; B01D 53/22 20060101 B01D053/22 |
Claims
1. A method for mitigating or preventing bubbles in a micro-fluidic
environment, comprising (a) degassing one or more of source
reagents prior to mixing of the source reagents; and (b) degassing
the mixed reagents prior to introducing the mixed reagents into the
micro-fluidic environment.
2. The method of claim 1, wherein the micro-fluidic environment is
a flow cell.
3. The method of claim 2, wherein the micro-fluidic environment is
a flow cell for the sequencing of a polynucleotide.
4. The method of claim 1, wherein the source reagents comprise 2,
3, 4, 5, 6, 7, or 8 different source reagents.
5. The method of claim 1 wherein degassing is achieved by passing
source fluid to be degassed through a gas-permeable tubing from
which gas present in the passing fluid is removed from the fluid
through the gas-permeable tubing into a vacuumed chamber or
space.
6. The method of claim 5, wherein the gas-permeable tubing is
stationed in a vacuumed chamber.
7. The method of claim 5, wherein the gas-permeable tubing is
encased in a vacuumed sheath that encases the gas-permeable tubing
along the length of the gas-permeable portion.
8. An apparatus comprising a degassing system performing a method
of any of claims 1-7.
9. An apparatus comprising a micro-fluidic device coupled to an
in-line degas system prior to the entrance of source reagents or
mixtures thereof into the micro-fluidic device.
10. The apparatus of claim 9, wherein the micro-fluidic device
comprises a flow cell.
11. The apparatus of claim 10, wherein the micro-fluidic device
comprises a flow cell for the sequencing of a polynucleotide.
12. The apparatus of claim 9, wherein the source reagents comprise
2, 3, 4, 5, 6, 7, or 8 different source reagents.
13. The apparatus of claim 9 wherein degassing is achieved by
passing source fluid to be degassed through a gas-permeable tubing
from which gas present in the passing fluid is removed from the
fluid through the gas-permeable tubing into a vacuumed chamber or
space.
14. The apparatus of claim 13, wherein the gas-permeable tubing is
stationed in a vacuumed chamber.
15. The apparatus of claim 13, wherein the gas-permeable tubing is
encased in a vacuumed sheath that encases the gas-permeable tubing
along the length of the gas-permeable portion.
16. A method for improving a chemical reaction in a micro-fluidic
environment, comprising removing bubbles from pre-mixed reagents or
mixed reagents prior to the pre-mixed or mixed reagents entering
the micro-fluidic environment.
17. The method of claim 16, comprising using any method or
apparatus of any of claims 1-15.
18. The method of claim 16, comprising removing bubbles from the
pre-mixed reagents and the mixed reagents prior to the mixed
reagents entering the micro-fluidic environment.
19. The method of claim 16, comprising removing bubbles from the
mixed reagents prior to such mixed reagents entering the
micro-fluidic environment along substantially the full length of
the connecting line between reagent mixing and a micro-fluidic
chemical system.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of and priority to U.S.
provisional application No. 61/034,140, filed Mar. 5, 2008, the
entirety of which is hereby incorporated herein by reference for
all purposes.
FIELD OF THE INVENTION
[0002] The invention generally relates to methods, systems and
devices for mitigation of bubbles in a micro-fluidic environment.
More particularly, the invention relates to methods, systems and
devices for mitigation of bubbles from reagents, solvents,
formulations and for improving chemical reactions in micro-fluidic
systems, such as for fluorescence detection and polynucleotide
sequencing.
BACKGROUND OF THE INVENTION
[0003] Recently, methods and apparatus for analyzing polynucleotide
sequences have been developed. See, e.g., U.S. Pat. No. 7,282,337;
U.S. Pat. No. 7,279,563; U.S. Pat. No. 7,226,720; U.S. Pat. No.
7,220,549; U.S. Pat. No. 7,169,560; U.S. Pat. No. 6,818,395; U.S.
Pat. No. 6,911,345; US Pub. Nos. 2006/0252077; 2007/0070349; and
2007-0070349. These automated methods and apparatus provide for
high speed and high throughput analysis of long polynucleotide
sequences with simplicity, flexibility and lower cost. See, e.g.,
www.helicosbio.com/, particularly information on HeliScope.TM.
Sequencer (e.g.,
www.helicosbio.com/Products/HelicosGeneticAnalysissystem/HeliScopetradeSe-
quencer/tabid/8 7/Default.aspx website visited as of Mar. 5,
2008).
[0004] In such methods and apparatus, and other related or
unrelated micro-fluidic devices or environments, it is sometimes
critical to have undesirable bubbles or gasses removed from the
operating fluids within the systems, such as from reagents,
buffers, solvents, etc. In general, in micro-fluidic devices
bubbles are a source of major problems, from inconsistent chemistry
to imaging problems and mechanical blockages. Inaccurate metering
of fluids or materials can degrade the precision and accuracy of
measurements. Gas bubbles can also interfere with the chemical or
physical properties or performance of the micro-fluidic system.
Thus, it is highly desirable that micro-fluidic devices, such as
employed in the automated polynucleotide sequencers, bubbles from
reagents, formulations and other fluids within the operating system
are reduced, minimized or eliminated.
SUMMARY OF THE INVENTION
[0005] The invention is based, in part, on the discovery that
degassing using gas-permeable tubing (e.g., in-line degassing)
contained in a vacuum chamber or a vacuumed sheath (or encase) can
effectively achieve the desired objective of reduction, minimizing
or elimination of bubbles in the down-stream micro-fluidic device
(for example, the flow cell in a polynucleotide sequencer).
[0006] In one aspect, the invention generally relates to a method
for mitigating or preventing bubbles in a micro-fluidic
environment. The method includes the steps of (a) degassing one or
more of source reagents prior to mixing of the source reagents; and
(b) degassing the mixed reagents prior to introducing the mixed
reagents into the micro-fluidic environment. In some embodiments of
the invention, the micro-fluidic environment is or comprises a flow
cell, such as a flow cell for the sequencing of a polynucleotide as
found in an apparatus or system for single molecular sequencing of
DNAs, RNAs or other poly- or oligo-nucleotides. The source reagents
may include 2, 3, 4, 5, 6, 7, 8 or more different reagents.
Degassing may be achieved by passing source fluids to be degassed
through a gas-permeable tubing from which gas present in the
passing fluid is removed from the fluid via the gas-permeable
tubing and into a vacuumed chamber or space. The gas-permeable
tubing may be stationed in an vacuumed chamber. In a preferred
embodiment, the gas-permeable tubing is encased in an vacuumed
sheath that encases the tubing along the length of the
gas-permeable portion.
[0007] In another aspect, the invention generally relates to an
apparatus that includes a micro-fluidic device fluidly connected to
an in-line degas system prior to the entrance of source reagents or
mixtures thereof into the micro-fluidic device. In some embodiments
of the invention, the micro-fluidic environment is or comprises a
flow cell, such as a flow cell for the sequencing of a
polynucleotide as found in an apparatus or system for single
molecular sequencing of DNA, RNA or other poly- or
oligo-nucleotides. The source reagents may include 2, 3, 4, 5, 6,
7, 8 or more different Teagents. Degassing may be achieved by
passing source fluids to be degassed through a gas-permeable tubing
from which gas present in the passing fluid is removed from the
fluid via the gas-permeable tubing and into a vacuumed chamber or
space. The gas-permeable tubing may be stationed in an vacuumed
chamber. In a preferred embodiment, the gas-permeable tubing is
encased in an vacuumed sheath that encases the tubing along the
length of the gas-permeable portion.
[0008] In another aspect, the invention generally relates to a
method for improving chemical reaction in a micro-fluidic
environment. The method includes removing bubbles from pre-mixed
reagents and mixed reagents prior to the mixed reagents entering
the micro-fluidic environment.
[0009] The foregoing aspects and embodiments of the invention may
be more fully understood by reference to the following figures,
detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] The invention may be further understood from the following
figures in which:
[0011] FIG. 1 is a schematic illustration of an embodiment of a
sequencing apparatus.
[0012] FIG. 2A is a schematic illustration of an embodiment of a
sequencing apparatus having at least one in-line degassing system
incorporated therein.
[0013] FIG. 2B is a schematic illustration of an embodiment of an
in-line degassing system coupled to a micro-fluidic device.
[0014] FIG. 3 is a schematic illustration of an embodiment of an
in-line degassing system.
[0015] FIG. 4A is a schematic illustration of an embodiment of an
in-line degassing systems.
[0016] FIG. 4B is a schematic illustration of an embodiment of an
in-line degassing systems.
DETAILED DESCRIPTION OF THE INVENTION
[0017] In its simplest sense, the invention relates to in-line
degassing using gas-permeable tubing contained in a vacuum chamber
or a vacuumed sheath (or encase) so as to effectively achieve the
desired objective of reduction, minimizing or elimination of
bubbles in the down-stream micro-fluidic device (for example, the
flow cell in a polynucleotide sequencer). The invention relates to
both the application of hardware to the described solution of the
presented problem of bubble mitigation in micro-fluidic devices and
a specific application technique to help eliminate bubbles from
micro-fluidic devices.
[0018] For example, as illustrated in FIG. 1, is a schematic
diagram of an embodiment of a sequencing apparatus. The apparatus
includes a plurality of reagents and/or buffer lines that are
fluidly connected to a multi-port valve and/or a mixing chamber
which output mixed solutions of the desired combination of reagents
to the down stream flow cell where reactions and/or detection or
imaging take place as designed.
[0019] In an improved system wherein an embodiment of the invention
is employed, as schematically illustrated in FIG. 2A, in-line
degassing using gas-permeable tubing contained in a vacuum chamber
or, preferably a vacuumed sheath (or encase) is used to reduce,
minimize or eliminate bubbles before the down-stream receiving
micro-fluidic device (for example, the flow cell in a
polynucleotide sequencer). For example, in FIG. 2A, each of the
incoming lines to the multi-port valve may be fitted with such
in-line degassing tubing (along with appropriate vacuumed chamber
or sheath). In addition, the outgoing line from the multi-port
valve to the flow cell may also be fitted with such in-line
degassing tubing (e.g., along the full length or substantially
along the full length of the connection line). In another example,
as schematically illustrated in FIG. 2B, four lines from four
reagent sources are directed to a syringe pump and mixed before
directed to the flow cell. A degassing line coupled to a vacuum
source can be incorporated so as to subject the exiting mixture
from the mixer to in-line degassing as the liquid travels to the
flow cell.
[0020] In a more general embodiment of the invention, as
schematically illustrated in FIG. 3, an in-line degassing system
includes a plurality of incoming lines to a fluid mixer, wherein
one or more of the incoming lines (e.g., all incoming lines) to the
fluid mixer are fitted with the degassing tubing along with the
corresponding vacuumed chamber or sheath. Alternative to,
optionally or in addition to the above, the outgoing line from the
fluid mixer to the down stream receiving micro-fluidic device is
fitted with the degassing tubing along with the corresponding
vacuumed chamber or sheath.
[0021] Some embodiments of the degassing system of the invention
are schematically illustrated in FIG. 4A and FIG. 4B, wherein a
tubing of gas-permeable material is encased in a vacuum chamber
(FIG. 4A) or preferably in an encased sheath along the length of
the gas permeable tubing (FIG. 4B). A pump source is connected to
the degassing line to create pressure differential and to remove
gasses of the bubbles. The gas-permeable tubing lets gas pass
through at pressure differentiation but does not allow liquid
fluids to permeate the tubing.
[0022] In one embodiment of the invention, as schematically
illustrated in FIG. 2B, the first step in removal of bubbles from a
closed chemistry system is to purge a first small amount of a
reaction formulation directly to waste, not through the
micro-fluidic device. This eliminates any bubbles at the top of the
syringe pump that are created by the mixing process or by the
increase in density which can occur in the mixing of organic
chemicals and inorganic chemicals.
[0023] In one example, the in-line degassing system used in an
automatic polynucleotide sequencer or analyzer consists of a coiled
tube made of gas permeable material contained inside a vacuum
chamber. As the fluid flows through the tube the dissolved gasses
are removed. This technique can be used to remove excess gasses
from organic solvents prior to their being mixed into a reagent
formulation (e.g., the vacuum chamber is in line between the
solvent bottle and the input to the liquid handling system such as
a mixer). This minimizes the out-gassing that results when organic
solvents, which can hold many times more gas than water, are mixed
with water. A similar chamber has been used on the output of the
system in-line with and before the micro-fluidic device (e.g., a
flow cell).
[0024] In another embodiment of the bubble removal system, a gas
permeable tube is contained in a sheath along its length (e.g.,
Rheodyne Part #PR100207A). It is the space between the tubing and
the sheath that is evacuated in this case. The length of tubing is
used as the output from the automated liquid system to the
micro-fluidic device. This works by removing dissolved gasses from
the final reagent formulation as it is pumped into the flow cell,
thus significantly reducing any out-gassing that may occur at
changes in pressure or temperature anywhere in the path to and
through the flow cell. The in-line version advantages include: no
increase in swept volume and decrease of cost of reagents. The
system also significantly reduces dispersion as the liquids are
moved into the flow cell since it has a much smaller internal
diameter.
[0025] This has been shown to reduce the occurrence of bubbles in
the flow cell from 6% of fluid transfers to less than 0.2% and
probably better.
[0026] One application of the invention, its technique and related
hardware, is its use in a sequencing system, e.g., the
HeliScope.TM. Single Molecule Sequencer (Helicos BioSciences
Corporation of Cambridge, Mass.) for automated creation of
formulations on the fly for single molecule sequencing. To achieve
its breakthrough performance, the sequencer incorporates a number
of advanced technologies and innovative engineering solutions:
[0027] Touch Screen Monitor & Graphical User Interface: A touch
screen monitor allows the user to interact with the instrument's
simple and flexible workflow driven interface to define and monitor
a run.
[0028] Integrated Barcode Readers: The system has integrated
barcode readers to assist in proper loading and tracking single
molecule sequencing reagents and flow cells.
[0029] Remote Web Tool: A web based tool allows the user remote
access and monitoring capabilities to the system to define runs,
download data and obtain run status from runs that are in
progress.
[0030] Precision Flow Cell: The instrument performs sequencing
reactions inside two precision flow cells. Alternating between the
two, it performs sequencing reactions in one while capturing images
from the other. The flow cells use a proprietary surface chemistry
to capture single molecules at a density of 100 million strands of
DNA per square centimeter. The current flow cell configuration
contains 25 discrete channels, enabling the analysis of up to 50
individual or multiplexed samples per run.
[0031] Fluid Delivery System: To maintain reagent integrity and
optimize performance across the duration of a single molecule
sequencing experiment, an advanced fluid delivery system provides
just-in-time reagent mixing and delivery to the flow cells.
[0032] High Speed Precision Stage: To capture images from the
single molecule sequencing reaction, the instrument incorporates a
high speed, thermally-controlled stage for accurate, repeatable
positioning during the imaging process.
[0033] System Monitoring: The sequencer provides real time
monitoring and alert capabilities of the system
environment--including reagent level sensing, temperature, pressure
and other critical operating parameters. All metrics are recorded
to a run log file for QC and traceability.
[0034] System UPS: The instrument contains its own uninterruptible
power supplies capable of allowing the instrument to reach a "safe"
stopping point in the sequencing-by-synthesis process.
Incorporation By Reference
[0035] The entire disclosure of each of the publications and patent
documents referred to herein is incorporated by reference in its
entirety for all purposes to the same extent as if each individual
publication or patent document were so individually denoted.
Equivalents
[0036] The invention may be embodied in other specific forms
without departure from the spirit or essential characteristics
thereof. The foregoing embodiments are therefore to be considered
in all respects illustrative rather than limiting on the invention
described herein. Scope of the invention is thus indicated by the
appended claims rather than by the foregoing description, and all
changes that come within the meaning and range of equivalency of
the claims are intended to be embraced therein.
* * * * *
References